1. Field of the Invention
The invention relates to a cancer screening method, and in particular, to a cancer screening method using methylated DNA as the biomarker.
2. Description of the Prior Art
Cervical cancer has been one of the main causes of death in females worldwide and in Taiwan. Based on the statistical survey by the World Health Organization (WHO) in 2002, cervical cancer was the second major disease responsible for the death of women worldwide, second to breast cancer. Regular cervical cancer screening is the best way to prevent cervical cancer. Conventional cervical cancer screening includes two approaches: the most commonly used Pap smear, and human papilloma virus testing (HPV testing). Pap smear consists of sampling secreta from cervix uteri, examining under microscope whether there is cancerous pathological change in the exfoliated epithelial cell, so as to detect cervical cancer early. HPV testing, on the other hand, relies on the detection of human papilloma virus (HPV) DNA.
There are, however, many undesired properties of Pap smear. For one, it requires sampling by a physician, and analysis by a medical examiner/pathologist, which is a high cost of manpower that poses difficulty on promoting the test in many developing countries. Also, Pap smear has a high false negative rate which delays diagnosis and proper treatment prior to cancerous pathological change. As for HPV testing, although it is highly sensitive, it tends to create a high false positive rate, which not only leaves patients worry in vain, but also wastes much medical resources in examinations follow up to those false positive patients. Accordingly, one of the important topics in promoting cervical cancer examination relies on increasing the accuracy and convenience of cervical cancer examination method.
Infection with oncogenic human papilloma virus (HPV) is the most significant risk factor in the etiology of cervical cancer. E6/E7 oncoprotein encoded by “high-risk” HPV types has been shown to interact with the tumor-suppressor gene p53/pRB, causing abnormal cell-cycle regulation (zur Hausen 2000). HPV DNA could be detected in virtually all cases of cervical cancers (Walboomers, Jacobs et al. 1999). However, HPV infection is necessary but not sufficient to cause cervical cancer. About 60% of LSIL (low-grade squamous intraepithelial lesion) regress, 30% persists, 5-10% progress to high-grade SIL (HSIL, or High-grade squamous intraepithelial lesion) and only less than 1% becomes cervical cancer (Syrjanen, Vayrynen et al. 1985; Syrjanen 1996). Persistence of HPV infection and viral load may be detrimental accounting the development of HSIL and cancer (Ylitalo, Sorensen et al. 2000). However, the molecular mechanism of cervical carcinogenesis remains illusive.
Other factors, such as environmental and genetic alterations, may also play a decisive role in malignant conversion of cervical keratinocytes (Magnusson, Sparen et al. 1999; Ylitalo, Sorensen et al. 1999). Despite initiation by HPV, genetic changes with resultant genomic instability has long been recognized as an important mechanism for cervical carcinogenesis. Cytogenetic studies have revealed non-random chromosomal changes in cervical cancers (Mitra, Rao et al. 1994; Atkin and Baker 1997; Harris, Lu et al. 2003). Several molecular genetic studies have identified a few frequent loss of heterozygosity (LOH) sites, suggesting the involvement of tumor suppressor genes (TSGs) in the development of cervical cancer. (Mitra, Murty et al. 1994; Mullokandov, Kholodilov et al. 1996; Rader, Kamarasova et al. 1996; Kersemaekers, Hermans et al. 1998; Mitra 1999).
Genomic deletions have long been considered to be an important factor in tumorigenesis. For a long time, we have been accustomed to the idea that the coding potential of the genome lies within the arrangement of the four A, T, G, C bases. The two-hit theory proposed as early as in 1970s indicates concomitant mutations or deletions of some homologous tumor suppressor genes may cause or predispose to cancer development (Knudson, Hethcote et al. 1975; Knudson 2001). However, additional information that affects phenotype can be stored in the modified base 5-methylcytosine. 5-Methylcytosine is found in mammals in the context of the palindromic sequence 5′-CpG-3′. Most CpG dinucleotide pairs are methylated in mammalian cells except some areas called “CpG island.” CpG islands are GC- and CpG-rich areas of approximately 1 kb, usually located in the vicinity of genes and often found near the promoter of widely expressed genes (Bird 1986; Larsen, Gundersen et al. 1992). Cytosine methylation occurs after DNA synthesis, by enzymatic transfer of a methyl group from the methyl donor S-adenosylmethionine to the carbon-5 position of cytosine. The enzymatic reaction is performed by DNA methyltransferases (DNMTs)(Laird 2003). DNMT1 is the main enzyme in mammals, and is responsible for the post-replicative restoration of hemi-methylated sites to full methylation, referred to as maintenance methylation, whereas DNMT3A and DNMT3B are thought to be involved primarily in methylating new sites, a process called de novo methylation (Okano, Bell et al. 1999; Robert, Morin et al. 2003).
Loss of methylation at CpG dinucleotides, i.e., general hypomethylation, was the first epigenetic abnormalities identified in cancer cells (Feinberg and Vogelstein 1983; Cheah, Wallace et al. 1984). However, during the past few years, it has become increasing apparent that site-specific hypermethylation, e.g., some tumor suppressor genes, is associated with loss of function which may provide selective advantages during carcinogenesis (Jones and Baylin 2002; Feinberg and Tycko 2004). Dense methylation of CpG islands at promoter regions can trigger chromatin remodeling through histone modifications with subsequent gene silencing (Geiman and Robertson 2002; Egger, Liang et al. 2004). Therefore, in addition to chromosomal deletions or genetic mutations, epigenetic silencing of tumor suppressor genes by promoter hypermethylation is commonly seen in human cancer (Baylin, Herman et al. 1998; Jones and Laird 1999; Baylin and Herman 2000).
Epidemiologic studies have recently shown the correlation of serum folate level, a major source of methyl group, with the infection and clearance of HPV (Piyathilake, Henao et al. 2004). Genetic polymorphisms of enzymes in the metabolism of methyl cycle were also reported to be associated with the development of cervical intraepithelial lesions (Henao, Piyathilake et al. 2004). As the concept of epigenetics evolves, studies exploring the association between DNA methylation and cervical cancer are also booming. Studies of DNA methylation in cervical cancer are accumulating, which showed the potential of using methylation as markers in cervical screening (Feng, Balasubramanian et al. 2005). With the nature of the interface between genetics and environment, the prevalence of methylation in tumor suppressor genes varies in different genes and different populations. The concept of methylator phenotypes with different disease behaviors was proposed with controversy. The methylator phenotype of cervical cancer and its interaction with HPV genotypes still remains unknown. The extent to which adenocarcinoma can be analogue to squamous cell carcinoma in terms of methylation patterns has never been investigated. What genes are specifically methylated in cervical cancer and how many genes are required to achieve clinical application will remain a blossoming issue in the coming future. The excavation of genes with higher contribution component to cervical carcinogenesis may shed light on the promise of using DNA methylation as a diagnostic marker as well as the development of a novel therapeutic intervention through epigenetic modulation.